Olig1 mini-promoters

ABSTRACT

Isolated polynucleotides comprising an OLIG1 promoter are provided, where an OLIG1 regulatory element is operably joined to an OLIG1 basal promoter utilizing a non-native spacing between the promoter and regulatory elements. The promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, etc.

FIELD OF THE INVENTION

The invention relates to gene promoters and regulatory elements. Morespecifically, the invention relates to novel OLIG1 promoter compositionsand related methods.

BACKGROUND

The Olig1 gene encodes a basic helix-loop-helix (bHLH) transcriptionfactor, OLIG1 that along with the OLIG2 regulates key stages of earlyoligodendrocyte development. Myelinating oligodendrocytes fulfilldiverse functional roles, such as ensheathment of neurons to facilitateelectrical conductivity, maintenance of axonal integrity andparticipation in signaling networks with neurons. Olig1 gene function iscritical for regulation of oligodendrogenesis during embryonic and fetalstages of CNS development, and it continues to be expressed in matureoligodendrocytes (Ligon et al. 2006). Elevated Olig1 expression has alsobeen found in certain brain tumors, particularly oligodendrogliomas(Hoang-Xuan et al. 2002).

There exists a significant need for promoter elements which are capableof driving expression in specific cell types and/or in specific regionsof the brain. Identification of minimal elements required for adequateexpression and specificity will allow ease of use in expressionconstructs.

SUMMARY OF THE INVENTION

The present invention provides novel nucleic acid sequence compositionsand methods, which relate to OLIG1 promoters having a sequence otherthan a native OLIG1 promoter.

In one embodiment of the invention, there is provided an isolatednucleic acid fragment comprising an OLIG1 mini-promoter, wherein theOLIG1 mini-promoter comprises one or more OLIG1 regulatory elementsoperably linked in a non-native conformation to an OLIG1 basal promoter.The OLIG1 promoter may have a nucleic acid sequence which issubstantially similar in sequence and function to SEQ ID NO: 1. The oneor more OLIG1 regulatory elements may have nucleic acid sequences whichare substantially similar in sequence and function to SEQ ID NO: 2, SEQID NO: 3, and/or SEQ ID NO: 4. The OLIG1 basal promoter may have anucleic acid sequence which is substantially similar in sequence andfunction to SEQ ID NO: 5. The OLIG1 promoter may further be operablylinked to an expressible sequence, e.g. reporter genes, genes encoding apolypeptide of interest, regulatory RNA sequences such as miRNA, siRNA,anti-sense RNA, etc., and the like. Reporter gene sequences include, forexample luciferase, beta-galactosidase, green fluorescent protein,enhanced green fluorescent protein, and the like as known in the art.The expressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expressible sequence may encode an RNA interference molecule.

In one embodiment, there is provided an expression vector comprising anOLIG1 mini-promoter element, wherein the OLIG1 mini-promoter comprisesone or more OLIG1 regulatory elements operably linked in a non-nativeconformation to an OLIG1 basal promoter. The OLIG1 promoter may have anucleic acid sequence which is substantially similar in sequence andfunction to SEQ ID NO: 1. The one or more OLIG1 regulatory elements mayhave nucleic acid sequences which are substantially similar in sequenceand function to SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. TheOLIG1 basal promoter may have a nucleic acid sequence which issubstantially similar in sequence and function to SEQ ID NO: 5. TheOLIG1 promoter may further be operably linked to an expressiblesequence, e.g. reporter genes, genes encoding a polypeptide of interest,regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., andthe like. Reporter gene sequences include, for example luciferase,beta-galactosidase, green fluorescent protein, enhanced greenfluorescent protein, and the like as known in the art. The expressiblesequence may encode a protein of interest, for example a therapeuticprotein, receptor, antibody, growth factor, and the like. Theexpressible sequence may encode an RNA interference molecule. Theexpression vector may further comprise a genomic targeting sequence. Thegenomic targeting sequence may be HPRT.

In one embodiment, there is provided a method for selective expressionof a gene, protein, RNA interference molecule or the like in a cell, themethod comprising introducing into the cell a expression vectorcomprising an OLIG1 mini-promoter element of the invention, wherein theOLIG1 mini-promoter element comprises an OLIG1 regulatory elementoperably linked in a non-native conformation to an OLIG1 basal promoterelement. Cells of interest include, without limitation, cells of theperipheral or central nervous system and progenitors thereof, e.g.embryonic stem cells, neural stem cells, neurons, glial cells,astrocytes, microgial cells, etc. The OLIG1 promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 1. The one or more OLIG1 regulatory elements may have nucleicacid sequences which are substantially similar in sequence and functionto SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. The OLIG1 basalpromoter may have a nucleic acid sequence which is substantially similarin sequence and function to SEQ ID NO: 5. The OLIG1 promoter may furtherbe operably linked to an expressible sequence, e.g. reporter genes,genes encoding a polypeptide of interest, regulatory RNA sequences suchas miRNA, siRNA, anti-sense RNA, etc., and the like. Reporter genesequences include, for example luciferase, beta-galactosidase, greenfluorescent protein, enhanced green fluorescent protein, and the like asknown in the art. The expressible sequence may encode a protein ofinterest, for example a therapeutic protein, receptor, antibody, growthfactor, and the like. The expressible sequence may encode an RNAinterference molecule. The expression vector may thus further comprise agenomic targeting sequence. The genomic targeting sequence may be HPRT.

In one embodiment of the invention, there is provided a method foridentifying or selectively labeling a cell, the method comprisingintroducing into the cell a expression vector comprising an OLIG1mini-promoter element operably linked to an expressible sequence,wherein the OLIG1 mini-promoter element comprises one or more OLIG1regulatory elements operably linked in a non-native conformation to anOLIG1 basal promoter element, and wherein the expressible sequencecomprises a reporter gene. The OLIG1 promoter may have a nucleic acidsequence which is substantially similar in sequence and function to SEQID NO: 1. The one or more OLIG1 regulatory elements may have nucleicacid sequences which are substantially similar in sequence and functionto SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. The OLIG1 basalpromoter may have a nucleic acid sequence which is substantially similarin sequence and function to SEQ ID NO: 5. In some embodiments, the cellis a peripheral or central nervous system cell or progenitors thereof,including, without limitation, embryonic stern cells, neural stem cells,oligodendrocytes, glial cells, astrocytes, neurons and the like.Reporter gene sequences include, for example luciferase,beta-galactosidase, green fluorescent protein, enhanced greenfluorescent protein, and the like as known in the art. The expressiblesequence may encode a protein of interest, for example a therapeuticprotein, receptor, antibody, growth factor, RNA interference moleculeand the like.

In one embodiment of the invention, there is provided a method formonitoring or tracking the development or maturation of a cell, themethod comprising: 1) introducing into the cell a expression vectorcomprising an OLIG1 mini-promoter element operably linked to anexpressible sequence, wherein the OLIG1 mini-promoter element comprisesone or more OLIG1 regulatory elements operably linked in a non-nativeconformation to an OLIG1 basal promoter element, and wherein theexpressible sequence comprises a reporter gene; and 2) detecting theexpression of the reporter gene in the progeny of the cell as a means ofdetermining the lineage, identity or developmental state of theprogenitor cell or progeny thereof. The OLIG1 promoter may have anucleic acid sequence which is substantially similar in sequence andfunction to SEQ ID NO: 1. The one or more OLIG1 regulatory elements mayhave nucleic acid sequences which are substantially similar in sequenceand function to SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. TheOLIG1 basal promoter may have a nucleic acid sequence which issubstantially similar in sequence and function to SEQ ID NO: 5. In someembodiments, the cell is a peripheral or central nervous system cell orprogenitors thereof, including, without limitation, embryonic stemcells, neural stem cells, oligodendrocytes, glial cells, neurons and thelike.

SHORT DESCRIPTION OF FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. DNA expression vector (pEMS1306) into which OLIG1 promoterelements were inserted for expression studies. The OLIG1 promoter with anucleic acid sequence corresponding to SEQ ID NO: 1 was inserted intothe multiple cloning site (MCS) of the pEMS1302 vector such that itbecame operably linked to the enhanced green fluorescent protein (EGFP)reporter gene. The final construct, called OLIG1-D, also contained theHPRT genomic targeting sequence, an ampicillin resistance gene (AmpR)for screening, and a transcriptional termination sequence (SV40 polyA),as well as other elements necessary for vector replication and geneexpression.

FIG. 2. From top to bottom, the human genomic sequence of OLIG1 locatedon chromosome 21 with an bent arrow indicating the transcription startsite, the gene exon as a black box, the non-coding conserved regions asaqua boxes with open black boxes defining our candidate regulatoryregions (basal promoter and regulatory elements), the conservationprofile between the human and mouse sequences with the grey areadelineating the 70% threshold used.

FIG. 3. EGFP expression is detected in multiple germline mice from theOLIG1-D mini-promoter strain in the initial anti-EGFPimmunocytochemistry screen. Figures A and B illustrate the expressionpattern throughout cortical (CTX) and subcortical (SC) regions followinganti-GFP immunocytochemistry. Within the cortex, individual cell bodiesare distinctly labelled and “puffy” processes surround these cells. TheEGFP expression does not appear to be enriched in white matter tracts orin the corpus callosum.

FIG. 4. Expression of reporter in oligodendrocytes by OLIG1-D promoterelement. The OLIG1-D DNA expression vector was introduced into mouseembryonic stem cells (ESCs) at the HPRT locus. The ESCs were used togenerate genetically modified mice containing OLIG1-D. Immunofluorescentanalysis of mouse brain tissue sections revealed EGFP reporterexpression in oligodendrocytes. Left—Anti-EGFP antibodies reveal diffusestaining (green) which partially overlaps with anti-olig2 staining foroligodendrocytes (red), and shows less staining in other cell types(blue, Toto3 nuclear stain). Right—Anti-EGFP antibodies reveal diffusestaining (green) which shows little overlap with neural specific (NeuN,red) staining and some overlap with general nuclear staining (blue,Toto3 nuclear stain).

DETAILED DESCRIPTION

The polynucleotide compositions of the present invention comprise anovel arrangement of OLIG1 promoter elements (also referred to herein asOLIG1 mini-promoters) as well as novel expression vectors comprisingsaid arrangement of OLIG1 promoter elements (or mini-promoters). Thepresent invention also includes various methods of utilizing these novelOLIG1 promoter (or mini-promoter) elements or expression vectors.

Provided is a sequence listing including certain of the OLIG1mini-promoters, wherein SEQ ID NO:1 comprises the human OLIG1mini-promoter (3042 bp). Nucleotides 1-1019 comprise the human OLIG1regulatory element 1, which corresponds to SEQ ID NO: 2. Nucleotides1020-1384 comprise the human OLIG1 regulatory element 2, whichcorresponds to SEQ ID NO: 3. Nucleotides 1385-2571 comprise the humanOLIG1 regulatory element 3, which corresponds to SEQ ID NO: 4.Nucleotides 2572-3042 comprise the human OLIG1 basal promoter element,which corresponds to SEQ ID NO: 5.

The term ‘OLIG1’ refers to the gene that encodes the OLIG1 protein, andincludes the controlling regulatory elements, e.g. promoters and thelike. The term ‘OLIG1’ refers to the gene which encodes the OLIG1protein, also referred to as oligodendrocyte transcription factor 1,oligodendrocyte-specific bHLH transcription factor 1, or BHLHB6. Thehuman homolog of OLIG1 is encoded by the human gene identified asEntrezGene #116448 and is located at chromosomal location 21q22.11. Theprotein encoded by human OLIG1 has the Protein Accession Q8TAK6.2(Swiss-Prot). Other mammalian OLIG1 homologs include but are not limitedto: Rattus norvegicus (EntrezGene #60394, Protein Accession #Q9WUQ3.2),Mus musculus (EntrezGene #50914, Protein Accession #Q9JKN5.2).

The term ‘promoter’ refers to the regulatory DNA region which controlstranscription or expression of a gene and which can be located adjacentto or overlapping a nucleotide or region of nucleotides at which RNAtranscription is initiated. A promoter contains specific DNA sequenceswhich bind protein factors, often referred to as transcription factors,which facilitate binding of RNA polymerase to the DNA leading to genetranscription. A ‘basal promoter’, also referred to as a ‘corepromoter’, generally refers to a promoter that contains all the basicnecessary elements to promote transcriptional expression of an operablylinked polynucleotide. Eukaryotic basal promoters typically, though notnecessarily, contain a TATA-box and/or a CAAT box. A ‘OLIG1 basalpromoter’, in the context of the present invention and as used herein,is a nucleic acid compound having a sequence with at least 65%, at least70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least99% similarity to SEQ ID NO: 5, and which comprises at least 1, usuallyat least 2, and may comprise all 3 of the identified conserved sequenceslisted in Table 1. The OLIG1 basal promoters of the present inventionmay comprise a TATA box such as that found in the native human OLIG1promoter and/or a CAAT box such as that found in the native human OLIG1promoter, and these elements should be positioned relative to thetranscriptional start site (+1) in a way that is reflective of thenative sequence.

TABLE 1 List of conserved sequences in the human OLIG1 basal promoter -SEQ ID NO: 5. The start and end coordinates of the sequences arerelative to the full SEQ ID NO: 5 sequence. Conservation determined byalignment of 28 vertebrate species available through the UCSC genomebrowser Start (relative End Invariant to SEQ ID NO: 5) (relative to SEQID NO: 5) sequence type 2838 2848 Conserved sequence 2911 2924 Conservedsequence 2980 2996 Conserved sequence

A promoter may also include one or more ‘regulatory elements’ which mayalso influence the expression or transcription by the promoter. Suchregulatory elements encode specific DNA sequences which bind otherfactors, which may include but are not limited to enhancers, silencers,insulators, and/or boundary elements. An ‘OLIG1 regulatory element’, inthe context of the present invention and as used herein, may be anucleic acid compound having a sequence with at least 65%, at least 70%,at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%similarity to SEQ ID NO: 2, and which comprises at least 4, usually atleast 6, and may comprise all 7 of the identified conserved sequenceslisted in Table 2. An OLIG1 regulatory element may alternately be anucleic acid compound having a sequence with at least 65%, at least 70%,at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%similarity to SEQ ID NO: 3, and which comprises at least 1, usually atleast 2, and may comprise all 3 of the identified conserved sequenceslisted in Table 3. An OLIG1 regulatory element may alternately be anucleic acid compound having a sequence with at least 65%, at least 70%,at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%similarity to SEQ ID NO: 4, and which comprises at least 3, usually atleast 4, and may comprise all 5 of the identified conserved sequenceslisted in Table 4. The present invention provides, in certainembodiments as described herein, different promoters of the OLIG1 gene.In some embodiments, the OLIG1 promoter comprises one or more OLIG1regulatory elements operably linked to a OLIG1 basal promoter. Incertain embodiments, the OLIG1 regulatory elements are directly joinedwith no intervening sequences. In other embodiments, the OLIG1regulatory elements may be operably linked with intervening sequences.In general the spacing between the regulatory elements is not more thanabout 15 KB, generally not more than about 10 KB, usually not more thanabout 1 KB, more often not more than about 500 nt, and may be not morethan about 100 nt, down to a direct joining of the two sequences.

TABLE 2 List of conserved sequences in the human OLIG1 regulatoryelement 1: SEQ ID NO: 2. The start and end coordinates of the sequencesare relative to the full SEQ ID NO: 2 sequence. Start (relative EndInvariant to SEQ ID NO: 2) (relative to SEQ ID NO: 2) sequence type 3748 Conserved sequence 89 110 Conserved sequence 138 164 Conservedsequence 184 259 Conserved sequence 288 661 Conserved sequence 683 857Conserved sequence 880 892 Conserved sequence

TABLE 3 List of conserved sequences in the human OLIG1 regulatoryelement 2: SEQ ID NO: 3. The start and end coordinates of the sequencesare relative to the full SEQ ID NO: 3 sequence. Start (relative EndInvariant to SEQ ID NO: 3) (relative to SEQ ID NO: 3) sequence type 10521070 Conserved sequence 1143 1164 Conserved sequence 1189 1231 Conservedsequence

TABLE 4 List of conserved sequences in the human OLIG1 regulatoryelement 3: SEQ ID NO: 4. The start and end coordinates of the sequencesare relative to the full SEQ ID NO: 4 sequence. Start (relative EndInvariant to SEQ ID NO: 4) (relative to SEQ ID NO: 4) sequence type 16221641 Conserved sequence 1695 2243 Conserved sequence 2299 2373 Conservedsequence 2384 2396 Conserved sequence 2420 2451 Conserved sequence

The term ‘operably linked’, in the context of the present invention,means joined in such a fashion as to work together to allowtranscription. In some embodiments of the invention, two polynucleotidesequences may be operably linked by being directly linked via anucleotide bond. In this fashion, the two operably linked elementscontain no intervening sequences and in being joined are able to directtranscription of an expression sequence. In other embodiments of theinvention, two elements may be operably linked by an interveningcompound, for instance a polynucleotide sequence of variable length. Insuch a fashion, the operably linked elements, although not directlyjuxtaposed, are still able to direct transcription of an expressionsequence. Thus, according to some embodiments of the invention, one ormore promoter elements may be operably linked to each other, andadditionally be operably linked to a downstream expression sequence,such that the linked promoter elements are able to direct expression ofthe downstream expression sequence.

The term ‘mini-promoter’ refers to a promoter in which certain promoterelements are combined in a non-native conformation, usually in such afashion as to reduce the overall size of the promoter compared to thenative conformation. For example, after identification of criticalpromoter elements, using one or more of various techniques, the nativesequences that intervene the identified elements may be partially orcompletely removed. Other non-native sequences may optionally beinserted between the identified promoter elements. A mini-promoter mayprovide certain advantages over native promoter conformations. Forexample, the smaller size of the mini-promoter may allow easier geneticmanipulation, ie. for the design and/or construction of expressionvectors or other recombinant DNA constructs. In addition, the smallersize may allow easier insertion of DNA constructs into host cells and/orgenomes, ie. via transfection, transformation, etc. Other advantages ofmini-promoters would be apparent to one of skill in the art. In someembodiments of the invention, there are thus provided novel OLIG1mini-promoters comprising one or more OLIG1 regulatory elements operablylinked in a non-native conformation to an OLIG1 basal promoter. Ingeneral the spacing between the one or more OLIG1 regulatory elementsand the OLIG1 basal promoter is not more than about 15 KB, generally notmore than about 10 KB, usually not more than about 1 KB, more often notmore than about 500 nt, and may be not more than about 100 nt, down to adirect joining of the two sequences.

The term ‘expressible sequence’ refers to a polynucleotide that isoperably linked to a promoter element, such that the promoter elementcauses transcriptional expression of the expression sequence. Anexpressible sequence is typically linked downstream, on the 3′-end ofthe promoter element(s) in order to achieve transcriptional expression.The result of this transcriptional expression is the production of anRNA macromolecule. The expressed RNA molecule may encode a protein andmay thus be subsequently translated by the appropriate cellularmachinery to produce a polypeptide protein molecule. In some embodimentsof the invention, the expression sequence may encode a reporter protein.Alternately, the RNA molecule may be an antisense, RNAi or othernon-coding RNA molecule, which may be capable of modulating theexpression of specific genes in a cell, as is known in the art.

The term ‘RNA’ as used in the present invention includes full-length RNAmolecules, which may be coding or non-coding sequences, fragments, andderivatives thereof. For example, a full-length RNA may initiallyencompass up to about 20 Kb or more of sequence, and frequently will beprocessed by splicing to generate a small mature RNA. Fragments, RNAi,miRNA and anti-sense molecules may be smaller, usually at least about 18nt. in length, at least about 20 nt in length, at least about 25 nt. inlength, and may be up to about 50 nt. in length, up to about 100 nt inlength, or more. RNA may be single stranded, double stranded, synthetic,isolated, partially isolated, essentially pure or recombinant. RNAcompounds may be naturally occurring, or they may be altered such thatthey differ from naturally occurring RNA compounds. Alterations mayinclude addition, deletion, substitution or modification of existingnucleotides. Such nucleotides may be either naturally occurring, ornon-naturally occurring nucleotides. Alterations may also involveaddition or insertion of non-nucleotide material, for instance at theend or ends of an existing RNA compound, or at a site that is internalto the RNA (ie. between two or more nucleotides).

The term ‘nucleic acid’ as used herein includes any nucleic acid, andmay be a deoxyribonucleotide or ribonucleotide polymer in either singleor double-stranded form. A ‘polynucleotide’ or ‘nucleotide polymer’ asused herein may include synthetic or mixed polymers of nucleic acids,both sense and antisense strands, and may be chemically or biochemicallymodified or may contain non-natural or derivatized nucleotide bases, aswill be readily appreciated by those skilled in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), and modified linkages (e.g.,alpha anomeric polynucleotides, etc.). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions.

A ‘purine’ is a heterocyclic organic compound containing fusedpyrimidine and imidazole rings, and acts as the parent compound forpurine bases, adenine (A) and guanine (G). ‘Nucleotides’ are generally apurine (R) or pyrimidine (Y) base covalently linked to a pentose,usually ribose or deoxyribose, where the sugar carries one or morephosphate groups. Nucleic acids are generally a polymer of nucleotidesjoined by 3′ 5′ phosphodiester linkages. As used herein ‘purine’ is usedto refer to the purine bases, A and G, and more broadly to include thenucleotide monomers, deoxyadenosine-5′-phosphate anddeoxyguanosine-5′-phosphate, as components of a polynucleotide chain. A‘pyrimidine’ is a single-ringed, organic base that forms nucleotidebases, such as cytosine (C), thymine (T) and uracil (U). As used herein‘pyrimidine’ is used to refer to the pyrimidine bases, C, T and U, andmore broadly to include the pyrimidine nucleotide monomers that alongwith purine nucleotides are the components of a polynucleotide chain.

It is within the capability of one of skill in the art to modify thesequence of a promoter nucleic acid, e.g. the provided basal promoterand regulatory sequences, in a manner that does not substantially changethe activity of the promoter element, i.e. the transcription rate of anexpressible sequence operably linked to a modified promoter sequence isat least about 65% the transcription rate of the original promoter, atleast about 75% the transcription rate of the original promotersequence, at least about 80%, at least about 90%, at least about 95%, atleast about 99%, or more in a selected cell or suitable in vitroenvironment. Such modified sequences would be considered to be‘functionally similar’ or to have ‘functional similarity’ or‘substantial functional similarity’ to the unmodified sequence. Suchmodifications may include insertions, deletions which may be truncationof the sequence or internal deletions, or substitutions. The level ofsequence modification to an original sequence will determine the‘sequence similarity’ of the original and modified sequences.Modification of the promoter elements of the present invention in afashion that does not significantly alter transcriptional activity, asdescribed above would result in sequences with ‘substantial sequencesimilarity’ to the original sequence i.e. the modified sequence has anucleic acid composition that is at least about 65% similar to theoriginal promoter sequence, at least about 75% similar to the originalpromoter sequence, at least about 80%, at least about 90%, at leastabout 95%, at least about 99%, or more similar to the original promotersequence. Thus, mini-promoter elements which have substantial functionaland/or sequence similarity are herein described and are within the scopeof the invention.

An ‘RNA interference molecule’, or ‘RNA interference sequence’ asdefined herein, may include, but is not limited to, an antisense RNAmolecule, a microRNA molecule or a short hairpin RNA (shRNA) molecule.Typically, RNA interference molecules are capable of target-specificmodulation of gene expression and exert their effect either by mediatingdegradation of the mRNA products of the target gene, or by preventingprotein translation from the mRNA of the target gene. The overall effectof interference with mRNA function is modulation of expression of theproduct of a target gene. This modulation can be measured in ways whichare routine in the art, for example by Northern blot assay or reversetranscriptase PCR of mRNA expression, Western blot or ELISA assay ofprotein expression, immunoprecipitation assay of protein expression,etc.

An ‘antisense RNA molecule’, as used herein, is typically a singlestranded RNA compound which binds to complementary RNA compounds, suchas target mRNA molecules, and blocks translation from the complementaryRNA compounds by sterically interfering with the normal translationalmachinery. Specific targeting of antisense RNA compounds to inhibit theexpression of a desired gene may design the antisense RNA compound tohave a homologous, complementary sequence to the desired gene. Perfecthomology is not necessary for inhibition of expression. Design of genespecific antisense RNA compounds, including nucleotide sequenceselection and additionally appropriate alterations, are known to one ofskill in the art.

The term ‘microRNA molecule’, ‘microRNA’ or ‘miRNA’, as used herein,refers to single-stranded RNA molecules, typically of about 21-23nucleotides in length, which are capable of modulating gene expression.Mature miRNA molecules are partially complementary to one or moremessenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. Without being bound by theory, miRNAs arefirst transcribed as primary transcripts or pri-miRNA with a cap andpoly-A tail and processed to short, 70-nucleotide stem-loop structuresknown as pre-miRNA in the cell nucleus. This processing is performed inanimals by a protein complex known as the Microprocessor complex,consisting of the nuclease Drosha and the double-stranded RNA bindingprotein Pasha. These pre-miRNAs are then processed to mature miRNAs inthe cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC).When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end. The remaining strand, knownas the anti-guide or passenger strand, is degraded as a RISC complexsubstrate. After integration into the active RISC complex, miRNAs basepair with their complementary mRNA molecules and induce mRNA degradationby argonaute proteins, the catalytically active members of the RISCcomplex. Animal miRNAs are usually complementary to a site in the 3′ UTRwhereas plant miRNAs are usually complementary to coding regions ofmRNAs.

The term ‘short hairpin RNA’ or ‘shRNA’ refers to RNA molecules havingan RNA sequence that makes a tight hairpin turn that can be used tosilence gene expression via RNA interference. The shRNA hairpinstructure is cleaved by the cellular machinery into siRNA, which is thenbound to the RNA-induced silencing complex (RISC). This complex binds toand cleaves mRNAs which match the siRNA that is bound to it. shRNA istranscribed by RNA Polymerase III whereas miRNA is transcribed by RNAPolymerase II. Techniques for designing target specific shRNA moleculesare known in the art.

An ‘expression vector’ is typically a nucleic acid molecule which is maybe integrating or autonomous, (i.e. self-replicating), and whichcontains the necessary components to achieve transcription of anexpressible sequence in a target cell, when introduced into the targetcell. Expression vectors may include plasmids, cosmids, phage, YAC, BAC,mini-chromosomes, viruses, e.g. retroviruses, adenovirus, lentivirus,SV-40, and the like; etc. Many such vectors have been described in theart and are suitable for use with the promoters of the presentinvention. Expression vectors of the present invention include apromoter as described herein, operably linked to an expressiblesequence, which may also be optionally operably linked to atranscription termination sequence, such as a polyadenylation sequence.The expression vector optionally contains nucleic acid elements whichconfer host selectivity, elements that facilitate replication of thevector, elements that facilitate integration of the vector into thegenome of the target cell, elements which confer properties, for exampleantibiotic resistance, to the target cell which allow selection orscreening of transformed cells and the like. Techniques and methods fordesign and construction of expression vectors are well known in the art.

It may be desirable, when driving expression of an expressible sequencewith a particular promoter system, to have the expression occur in astable and consistent manner. A factor that has been shown to affectexpression is the site of integration of an expression vector orconstruct into the genome of the target cell, sometimes called ‘positioneffects’. Such position effects may be caused by, for example, localchromatin structure which affects expression of sequences from thatregion of the genome. One method to control for position effects whenintegrating an expression vector or construct into the genome of atarget cell is to include a ‘genomic targeting sequence’ in the vectoror construct that directs integration of the vector or construct to aspecific genomic site. As an example, the hypoxanthinephosphoribosyltransferase (HPRT) gene has been used successfully forthis purpose (Bronson et. al. 1996; Jasin et al. 1996). The HPRT genehas additional advantages as a genomic targeting sequence, for instanceits concomitant use as a selectable marker system. Other genomictargeting sequences that may be useful in the present invention aredescribed in the art, for instance (Jasin et al. 1996; van der Weyden etal. 2002). The genomic targeting signals as described herein are usefulin certain embodiments of the present invention.

Introduction of nucleic acids or expression vectors may be accomplishedusing techniques well known in the art, for example microinjection,electroporation, particle bombardment, or chemical transformation, suchas calcium-mediated transformation, as described for example in Maniatiset al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring HarborLaboratory or in Ausubel et al. 1994, Current protocols in molecularbiology, Jolm Wiley and Sons.

OLIG1 Promoters

The present invention herein provides novel OLIG1 mini-promotersequences which are capable of effecting transcriptional expression in aspatial and temporal fashion similar to naturally occurring OLIG1promoters. The OLIG1 mini-promoters of the invention comprise OLIG1promoter elements joined in a non-native configuration, thus providingadvantageous characteristics. Also provided are novel expression vectorcompositions comprising OLIG1 mini-promoters which allow consistentspecific spatiotemporal transcription of expression sequences. Alsoprovided are novel methods utilizing these OLIG1 mini-promoters andexpression vectors.

The OLIG1 promoters of the invention, as described herein, are referredto as ‘mini-promoters’ to reflect the fact that the mini-promoterscomprise OLIG1 promoter elements that are joined in a non-nativeconfiguration. In this context, the native intervening sequences mayhave been partially or completely removed, and optionally may have beenreplaced with non-native sequences. In such a fashion, the naturalspacing of the promoter elements, for instance the human OLIG1regulatory elements corresponding to SEQ ID NO: 2, SEQ ID NO: 3 and/orSEQ ID NO: 4 and the human OLIG1 basal promoter element corresponding toSEQ ID NO: 5, or sequences with substantial functional and/or sequenceequivalence, is altered. An advantage of such non-native mini-promotersis that the removal of native intervening sequences reduces the size ofthe mini-promoter while maintaining the functional activity of thepromoter, thus improving the utility of the mini-promoter for variousapplications.

The inventors have demonstrated, as illustrated in the non-limitingWorking Examples, that a human OLIG1 mini-promoter having a sequencecorresponding to SEQ ID NO: 1, and which is comprised of directly linkedhuman OLIG1 regulatory elements having a nucleic acid sequencecorresponding to SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4 operablylinked in a non-native conformation to a human OLIG1 basal promoterhaving a nucleic acid sequence corresponding to SEQ ID NO: 5, is capableof directing expression of an expressible sequence which is operablylinked downstream of the OLIG1 promoter in specific cell types indifferent regions of the brain. The OLIG1 regulatory elements (SEQ IDNOs: 2, 3, 4) and OLIG1 basal promoter element (SEQ ID NO: 5) havesequences which are identical to those found upstream of the human OLIG1gene, found on chromosome 21 of the human genome. To place thesesequences in context, SEQ ID NO: 2 corresponds to absolute genomiccoordinates chr21:33358409-33359425 (strand +); SEQ ID NO: 3 correspondsto absolute genomic coordinates chr21:33373177-33373543 (strand +); SEQID NO: 4 corresponds to absolute genomic coordinates chr21:33389511-33390692 (strand +); while SEQ ID NO: 5 corresponds to absolutegenomic coordinates chr:21 33363932-33364402 (strand +), where thegenomic coordinates are derived from NCBI Build 36.1 human genomeassembly of March 2006. It is within the skill of one in the art tolocate and determine these relative positions based on publishedsequence information for this gene, for instance found in the GenBank orPubMed public databases. It is understood that these genomic coordinatesand relative positions are provided for the purposes of context, andthat if any discrepancies exist between published sequences and thesequence listings provided herein, then the sequence listings shallprevail.

Promoters of the present invention may be modified with respect to thenative regulatory and/or native basal promoter sequence. In general,such modifications will not change the functional activity of thepromoter with respect to cell-type selectivity; and to the rate oftranscription in cells where the promoter is active. The modifiedpromoter provide for a transcription rate in a cell of interest of anexpressible sequence operably linked to a modified promoter sequencethat is at least about 75% the transcription rate of the promotersequence of SEQ ID NO:1, at least about 80%, at least about 90%, atleast about 95%, at least about 99%, or more. Methods of assessingpromoter strength and selectivity are known in the art, including, forexample, expression of a reporter sequence in a cell in vivo or invitro, and quantitating the reporter activity.

Modifications of interest include deletion of terminal or internalregions, and substitution or insertion of residues. Applicants haveidentified 18 conserved sequences in the OLIG1 promoter (Table 5), where12 such conserved sequences are present in the regulatory element, and 6conserved sequences are present in the basal promoter. A promoter ofinterest in the present invention comprises generally at least 5, atleast 10, usually at least 15, and may comprise all 18 of the identifiedconserved sequences, where the arrangement and spacing of conservedsequences may be the same as the native regulatory sequence, or may bealtered, e.g. in the positioning and spacing of elements. Sequences setforth in SEQ ID NO:1 that are not conserved may be deleted orsubstituted, usually modifications that retain the spacing betweenconserved sequences is preferred. In general the spacing between each ofthe regulatory elements and between the regulatory elements and thebasal promoter is not more than about 10 KB, generally not more thanabout 1 KB, usually not more than about 500 nt, and may be not more thanabout 100 nt, down to a direct joining of the two sequences.

TABLE 5 List of conserved sequences in SEQ ID NO: 1 (basal promoter + 3regulatory elements). The start and end coordinates of the sequences arerelative to the full SEQ ID NO: 1 sequence. Start (relative EndInvariant to SEQ ID NO: 1) (relative to SEQ ID NO: 1) sequence type 3748 Conserved sequence 89 110 Conserved sequence 138 164 Conservedsequence 184 259 Conserved sequence 288 661 Conserved sequence 683 857Conserved sequence 880 892 Conserved sequence 1052 1070 Conservedsequence 1143 1164 Conserved sequence 1189 1231 Conserved sequence 16221641 Conserved sequence 1695 2243 Conserved sequence 2299 2373 Conservedsequence 2384 2396 Conserved sequence 2420 2451 Conserved sequence 28382848 Conserved sequence 2911 2924 Conserved sequence 2980 2996 Conservedsequence

In some embodiments of the invention, there is thus provided an isolatednucleic acid fragment comprising an OLIG1 mini-promoter, wherein theOLIG1 promoter comprises an OLIG1 regulatory element operably linked ina non-native conformation to an OLIG1 basal promoter. In certainembodiments of the invention, the OLIG1 promoter may have a nucleic acidsequence which is substantially similar in sequence and function to SEQID NO: 1. The one or more OLIG1 regulatory elements may have nucleicacid sequences which are substantially similar in sequence and functionto SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. The OLIG1 basalpromoter may have a nucleic acid sequence which is substantially similarin sequence and function to SEQ ID NO: 5. The OLIG1 promoter may furtherbe operably linked to an expressible sequence, e.g. reporter genes,genes encoding a polypeptide of interest, regulatory RNA sequences suchas miRNA, siRNA, anti-sense RNA, etc., and the like. Reporter genesequences include, for example luciferase, beta-galactosidase, greenfluorescent protein, enhanced green fluorescent protein, and the like asknown in the art. The expressible sequence may encode a protein ofinterest, for example a therapeutic protein, receptor, antibody, growthfactor, and the like.

It is an object of the present invention to provide means of expressinga gene, protein, RNA interference molecule or the like in a cell, tissueor organ. As such, the inventors thus provide novel expression vectorscomprising OLIG1 mini-promoters which are capable of accomplishing thistask. In some embodiments of the invention, there is provided aexpression vector comprising an OLIG1 promoter element, wherein theOLIG1 promoter element comprises an OLIG1 regulatory element operablylinked in a non-native conformation to an OLIG1 basal promoter element.The OLIG1 promoter element may have a nucleic acid sequencesubstantially similar in sequence and function to SEQ ID NO: 1. TheOLIG1 regulatory element may have a nucleic acid sequence substantiallysimilar in sequence and function to SEQ ID NO: 2. The OLIG1 basalpromoter element may have a nucleic acid sequence substantially similarin sequence and function to SEQ ID NO: 3. The OLIG1 promoter may furtherbe operably linked to an expressible sequence, e.g. reporter genes,genes encoding a polypeptide of interest, regulatory RNA sequences suchas miRNA, siRNA, anti-sense RNA, etc., and the like. Reporter genesequences include, for example luciferase, beta-galactosidase, greenfluorescent protein, enhanced green fluorescent protein, and the like asknown in the art. The expressible sequence may encode a protein ofinterest, for example a therapeutic protein, receptor, antibody, growthfactor, and the like. The expression vector may further comprise agenomic targeting sequence. The genomic targeting sequence may be HPRT.

It is an object of the present invention to provide means of expressinga gene, protein, RNA interference molecule or the like in a cell, tissueor organ. As such, the inventors thus provide novel expression vectorscomprising OLIG1 mini-promoters which are capable of accomplishing thistask. In some embodiments of the invention, there is provided anexpression vector comprising an OLIG1 promoter element, wherein theOLIG1 promoter element comprises one or more OLIG1 regulatory elementsoperably linked in a non-native conformation to an OLIG1 basal promoterelement. The OLIG1 promoter element may have a nucleic acid sequencesubstantially similar in sequence and function to SEQ ID NO: 1. The oneor more OLIG1 regulatory elements may have nucleic acid sequences whichare substantially similar in sequence and function to SEQ ID NO: 2, SEQID NO: 3, and/or SEQ ID NO: 4. The OLIG1 basal promoter may have anucleic acid sequence which is substantially similar in sequence andfunction to SEQ ID NO: 5. The OLIG1 promoter may further be operablylinked to an expressible sequence, e.g. reporter genes, genes encoding apolypeptide of interest, regulatory RNA sequences such as miRNA, siRNA,anti-sense RNA, etc., and the like. Reporter gene sequences include, forexample luciferase, beta-galactosidase, green fluorescent protein,enhanced green fluorescent protein, and the like as known in the art.The expressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expression vector may further comprise a genomic targeting sequence.The genomic targeting sequence may be HPRT.

The inventors have herein demonstrated that expression vectorscomprising novel OLIG1 mini-promoter elements are capable of directingtranscription of an expression sequence in specific cell types inspecific regions of the brain, most notably the cortical and subcorticalregions of the brain. In some embodiments of the invention, there isthus provided a method for expressing a gene, protein, RNA interferencemolecule or the like in the targeted cells of the brain. Cells ofinterest include, without limitation, cells of the peripheral or centralnervous system and progenitors thereof, e.g. embryonic stem cells,neural stem cells, neurons, oligodendrocytes, glial cells, astrocytes,microgial cells, etc. The method comprises introducing into a cell orprogenitor cell thereof an expression vector comprising an OLIG1mini-promoter element, wherein the OLIG1 mini-promoter element comprisesone or more OLIG1 regulatory elements operably linked in a non-nativeconformation to an OLIG1 basal promoter element. The OLIG1 promoterelement may have a nucleic acid sequence substantially similar insequence and function to SEQ ID NO: 1. The one or more OLIG1 regulatoryelements may have nucleic acid sequences which are substantially similarin sequence and function to SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ IDNO: 4. The OLIG1 basal promoter may have a nucleic acid sequence whichis substantially similar in sequence and function to SEQ ID NO: 5. TheOLIG1 promoter may further be operably linked to an expressiblesequence, e.g. reporter genes, genes encoding a polypeptide of interest,regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., andthe like. Reporter gene sequences include, for example luciferase,beta-galactosidase, green fluorescent protein, enhanced greenfluorescent protein, and the like as known in the art. The expressiblesequence may encode a protein of interest, for example a therapeuticprotein, receptor, antibody, growth factor, and the like. The expressionvector may thus further comprise a genomic targeting sequence. Thegenomic targeting sequence may be HPRT.

In other embodiments of the invention, there is provided a method foridentifying or labeling a cell, the method comprising introducing intothe cell a expression vector comprising an OLIG1 mini-promoter elementoperably linked to an expressible sequence, wherein the OLIG1mini-promoter element comprises an OLIG1 regulatory element operablylinked in a non-native conformation to an OLIG1 basal promoter element,and wherein the expressible sequence comprises a reporter gene. TheOLIG1 promoter element may have a nucleic acid sequence substantiallysimilar in sequence and function to SEQ ID NO: 1. The one or more OLIG1regulatory elements may have nucleic acid sequences which aresubstantially similar in sequence and function to SEQ ID NO: 2, SEQ IDNO: 3, and/or SEQ ID NO: 4. The OLIG1 basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 5. The inventors have demonstrated that expression vectorscomprising certain human OLIG1 promoter elements are capable ofexpression in specific regions of the brain, most notably the corticaland subcortical regions of the brain. In some embodiments, the cell is aperipheral or central nervous system cell or progenitors thereof,including, without limitation, embryonic stem cells, neural stem cells,oligodendrocytes, glial cell, neuronal cells, astrocytes, and the like.Reporter gene sequences include, for example luciferase,beta-galactosidase, green fluorescent protein, enhanced greenfluorescent protein, and the like as known in the art. The expressiblesequence may encode a protein of interest, for example a therapeuticprotein, receptor, antibody, growth factor, RNA interference moleculeand the like.

In further embodiments of the invention, there is provided a method formonitoring or tracking the development or maturation of a cell,including a cell of neural or oligodendrocyte lineage. The methodcomprises: 1) introducing into a progenitor to a cell, e.g. an embryonicstem cells, neural stem cell, neuronal progenitor cell, neuronal cell,oligodendrocyte progenitor cell, oligodendrocyte cell etc., anexpression vector comprising an OLIG1 mini-promoter element operablylinked to an expressible sequence, wherein the OLIG1 mini-promoterelement comprises one or more OLIG1 regulatory elements operably linkedin a non-native conformation to an OLIG1 basal promoter element, andwherein the expressible sequence comprises. a reporter gene; and 2)detecting the expression of the reporter gene in cell progeny of thecells as a means of determining the lineage, identity or developmentalstate of the cell or cell progeny. In such a fashion, one may be able tofollow the development of a parent cell as it differentiates into moremature cells. As an example, one could introduce a expression vectorcomprising the aforementioned OLIG1 promoter elements into a pluripotentstem cell, monitor the expression of the reporter gene that is beingexpressed by the OLIG1 promoter elements during the maturation anddifferentiation of the stem cell and thus determine the state ofmaturation, for instance in the differentiation of the pluripotent stemcell into a mature cell. The inventors have demonstrated that the OLIG1promoter elements described herein direct transcriptional expression incertain cell types, and so detection of reporter gene expression in acell would thus be indicative of the cellular identity of the cell asbeing a particular type of cell.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

WORKING EXAMPLES General Methods

Expression vector. The nucleic acid fragment corresponding to SEQ ID NO:1 was inserted into the multiple cloning site of the pEMS1306 (seeFIG. 1) to produce the expression vector OLIG1-D.

Derivation of mEMS1204 embryonic stem cells. Blastocysts were obtainedfrom natural mating of B6-Hprt1^(b-m3) females to 129-ROSA26 males at3.5 dpc. Blastocysts were flushed from uterine horns as per (Hogan etal. 1994), cultured in EmbryoMax® KSOM with ½ Amino Acids, Glucose andPhenol Red (Cat #MR-121, Millipore/Chermicon, Temecula, Calif.) for 3-5h, and then transferred onto mitomycin C (mitC; Cat#M4287, Sigma,Oakville, ON) mitotically inactivated B6-Hprt1^(b-m3), B6129F1, or 129mouse embryonic feeders (MEFs) derived from 13.5-day post-coital embryos(Ponchio et al. 2000) in 96-well plates containing KSR-ESC (Knockout™D-MEM, Cat#10829-018, Invitrogen, Burlington, ON) with 2 mM L-glutamine(Cat#25030-081, Invitrogen, Burlington, ON), 0.1 mM MEM nonessentialamino acid solution (Cat#11140-050, Invitrogen, Burlington, ON) and 16%Knockout™ Serum Replacement (Cat#10828-028, Invitrogen, Burlington, ON))media (MEF media was replaced 3-5 hour prior to transfer).

Blastocysts were cultured as per (Cheng et al. 2004) with the followingmodifications: Cells were cultured for 7-9 days in KSR-ESC with minimaldisturbance (checked on day 2 to determine if the blastocysts had‘hatched’ out of the zona pellucida) and no media changes. Blastocystswhich hatched and had a well developed ICM (inner cell mass) weretreated with 20 μl 0.25% trypsin-EDTA (Invitrogen, Burlington, ON) for 5min at 37° C., triturated with a 200 μl pipetman, inactivated with 30 μl0.5 mg/ml soybean trypsin inhibitor (Invitrogen, Burlington, ON), andbrought up to 200 μl with KSR-ESC, then transferred individually to a24-well MEF plate containing 1800 μl KSR-ESC, for a total volume of 2ml.

Beginning 4 days later, KSR-ESC media was replaced with FBS-ESC media(DMEM (Cat #11960-069, Invitrogen, Burlington, ON) with 2 mM L-glutamine(Invitrogen, Burlington, ON), 0.1 mM MEM nonessential amino acidsolution (Invitrogen, Burlington, ON), 16% ES Cell Qualified fetalbovine serum (FBS, Invitrogen, Burlington, ON) and 0.01%β-mercaptoethanol (Sigma, Oakville, ON) in 25%, 50%, 75% proportions(respectively) to adapt the cells to FBS containing media.

On day 7 the cells were trypsinized to one well of a 24 well platecontaining 1 ml of 100% FBS-ESC media, with daily media replacement.Once confluent, wells containing ESC colonies were expanded 3×24 wells(with MEFs), then passaged to 3×24 (with MEFs) and 3×12 well (plastic—noMEFs) for DNA analysis. Once confluent, the 3×24 wells were combined,aliquoted (3 vials), and frozen in ESC-freeze media (50% FBS, 40%FBS-ESC media, 10% DMSO (Sigma, Oakville, ON), and the 3×12 well treatedwith lysis buffer (Fisher Scientific, Ottawa, ON), mixed and aliquoted.Cultures were genotyped for X & Y chromosomes (Clapcote and Roder 2005),Gt(ROSA)26Sor^(tm1Sor) and WT alleles and Hprt1^(b-m3) and WT alleles.B6129F1-Gt(ROSA)26Sor^(tm1Sor)/+, Hprt1^(b-m3)/Y andB6129F1-Gt(ROSA)26Sor^(tm1Sor)+/+, Hprt1^(b-m3)/Y cell lines wereidentified.

Knock-in at the Hprt1 locus. The OLIG1-C plasmid DNA was purified withQiagen Maxi Kit (Qiagen, Mississauga, ON), resuspended in 10:1 Tris-EDTA(TE, pH7.0) buffer, and linearized with I-Scel (New England Biolabs,Pickering, ON). Linearized plasmid DNA was resuspended in 85 μl of TE(10:0.1) to a final concentration of 187.5 ng/μl. mEMS1204 ESCs weregrown to confluence on 4-6 T75 flasks of mitC treated Hprt1^(b-m3) mouseembryonic feeders (MEFs) in FBS-ESC media. ESCs (1.7−2.5×10⁷) in 720 μl1×PBS were added to the linearized DNA and electroporated in a 4 mmelectroporation cuvette (Bio-Rad Genepulser, Mississauga, ON), at 240 V,50 μF, 6-10 msec pulse, immediately resuspended in a total volume of 5ml of FBS-ESC media and plated onto 5×100 mm dishes of mitC B6129F1 MEFsin a total volume of 12 ml/100 mm dish. 24-36 h post-electroporation,correctly targeted homologous recombinants were selected for using HATmedia (FBS-ESC media containing 1×HAT ((0.1 mM sodium hypoxanthine, 0.4mM aminopterin, 0.16 mM thymidine), Cat#21060-017, Invitrogen,Burlington, ON). HAT media was changed every day for the first 3 days,and then every 3^(rd) day thereafter, for up to 10 days. Individualcolonies were counted and, typically, no more than 2 isolated colonieswere picked per 100 mm dish to optimize for independent homologousrecombination events. These colonies were expanded under standardprotocols for verification of the desired recombination event.

Derivation of knock-in mice. Chimeric mice from untargeted and targetedESCs were generated by microinjection (Hogan et al. 1994) into B6(E14TG2a derived) and B6-Alb (E14TG2a and mEMS1204 derived) E3.5blastocysts, or co-culture (Lee et al. 2007) with diploid ICR (CharlesRiver, Wilmington Wash. Stock#022) E2.5 morula (cultured overnight tothe blastocyst stage), followed by implantation into the uterine hornsof 2.5 day pseudopregnant ICR females. Chimeras were identified and coatcolor chimerism determined as outlined below.

Male chimeras derived from the E14TG2a cell lines were mated with B6 orB6-Alb females, and germline transmission was identified in the formercase by the transmission of the dominant A^(w) (nonagouti; white belliedagouti) allele, making the progeny appear brown with a cream belly, orin the later case by the combination of A^(w) and Tyr^(c-ch)(tyrosinase; chinchilla), making the progeny appear golden. Non-germlineprogeny from the cross to B6 were homozygous for the recessive a(nonagouti; nonagouti) allele and appeared black, whereas non-germlineprogeny from the cross to B6-Alb were homozygous for the recessiveTyr^(c) (tyrosinase; albino) allele and appeared white.

Male chimeras derived from the mEMS1204 cell lines were mated withB6-Alb females, and germline transmission identified by the presence ofthe dominant Tyr⁺ (tyrosinase; wild type) and the A^(w) (nonagouti;white bellied agouti) or a (nonagouti; nonagouti) alleles making theprogeny appear brown with a cream belly or black, respectively.Non-germline progeny were homozygous for the recessive Tyr^(c-2J)(tyrosinase; albino 2 Jackson) allele and appear white. All germlinefemale offspring should carry the knock-in X Chromosome and were matedwith B6 males. N2 offspring were analyzed for the presence of the KIallele by PCR.

Determination of coat color chimerism. E14TG2a- and mEMS1204-derivedchimeras were identified and level of coat color chimerism determined asfollows. E14TG2a ESCs, homozygous for A^(w) and Tyc^(c-ch) as they arederived from the 129/OlaHsd strain (Hooper et al. 1987a; Hooper et al.1987b), will produce chimeras with cream/chinchilla and agouti patcheson a black background when injected into B6 blastocysts. Thecream/chinchilla patches result from melanocytes derived solely from theESCs (A^(w)/A^(w), Tyr^(c-ch)/Tyr^(c-ch)), whereas agouti patches resultfrom melanocytes that are a mixture of ESC (A^(w)/A^(w),Tyr^(c-ch)/Tyr^(c-ch)) and host (a/a, Tyr^(c)/Tyr^(c)). However, E14TG2aESCs, when injected into B6-Alb (a/a, Tyr^(c)/Tyr^(c)) produce chimeraswith chinchilla and light chinchilla coat color patches on a whitebackground. The former is derived solely from the ESCs (A^(w)/A^(w),Tyr^(c-ch)/Tyr^(c-ch)), whereas the latter is again a mix of the ESC(A^(w)/A^(w), Tyr^(c-ch/)Tyr^(c-ch)) and host (a/a, Tyr^(c)/Tyr^(c)).mEMS1204-derived chimeras were identified and coat color chimerismdetermined in the same manner.

mEMS1204 ESCs, heterozygous A^(w)/a and homozygous for the wild typeTyr⁺ alleles will produce chimeras with agouti and black patches on awhite background when injected into B6-Alb blastocysts. The agoutipatches result from melanocytes derived solely from the ESCs (A^(w)/a,Tyr^(+/)Tyr⁺), whereas ‘black’ patches result from melanocytes that area mixture of ESC (A^(w)/a, Tyr^(+/)/Tyr⁺) and host (a/a,Tyr^(c-2J)/Tyr^(c-2J)).

For E14TG2a injections into B6 and mEMS1204 injections into B6-Alb,overall chimerism was calculated by summing the percent of coat colorpatches derived solely from the ESC, plus half the percent of theESC+host areas, where we conservatively estimated that half themelanocytes derive from the ESC and half from the host. For E14TG2ainjections into B6-Alb, the similarity between chinchilla and lightchinchilla on a white background presented difficulty when attempting toestimate overall coat color chimerism. As such, we estimated the percentchimerism based solely on the total chimerism observed when compared toa white mouse, resulting in slightly inflated overall percent chimerismfor this small cohort of mice.

Immunohistochemistry and Immunofluorescence. Adult male chimeric and agematched control mice were perfused with 4% paraformaldehyde (PFA) aspreviously described (Young et al. 2002). Whole brains were dissectedout and post-perfusion immersion fixed with PFA for 2-3 hours at 4° C.Brains were then transferred to 20% sucrose at 4° C. overnight withgentle shaking. The brains were cryostat sectioned sagittally at 12-14μm and mounted on superfost-plus slides (Cat# 12-550-15, ThermoFisherScientific, Waltham, Mass.). EGFP expression was detected by directfluorescence of EGFP or by indirect immunofluorescence with anti-GFPantibodies (Abcam, Cambridge, Mass.) using a BioRad confocal laserscanning microscope (CLSM, BioRad, Hercules, Calif.).

For double label immunofluorescence analyses to determine cell types inthe cerebellum, anti-GFAP was used in conjunction with direct EGFPfluorescence and imaged by CLSM (Liu et al. 2007). In brief, slidemounted brain sections, were permeabilized with phosphate bufferedsaline containing 0.1% triton-X100 (PBST), blocked with PBST containing5% normal horse serum and 1% BSA, then incubated with primary antibodiesovernight at room temperature in a humid chamber. Following three washeswith PBST, the tissue were incubated with secondary antibodies (goatanti-rabbit-Alexa-594 conjugate, Molecular Probes, Eugene, Oreg.). Theslides were counterstained with TOTO3/DAPI (1 μM each) for labeling allnuclei in confocal images. Bright field analyses were also conductedfollowing immunocytochemical detection of anti-GFP using the VectastainABC kit and DAB as the chromogen to give a brown reaction productfollowing the manufacturer's directions. Bright field images werevisualized on a Zeiss Axiovert microscope and Axiovision Software (CarlZeiss Microimaging, Thornwood, N.Y.).

Selection of OLIG1 promoter elements. Cross-species comparisons, orphylogenetic footprinting, were identified as a means to predictregulatory regions. The two mammalian species with the best evolutionarydistance to use this approach are human and mouse. In the specific caseof OLIG1, we computed the conservation level between human and mousetaking into consideration the non-coding sequence surrounding the OLIG1gene. This genomic region including a lot of non-coding sequencesconserved down to the frog, we set up a threshold of 70% of identity toselect our candidate regulatory regions (FIG. 2). The OLIG1 basalpromoter (SEQ ID NO: 5) and three regulatory regions (SEQ ID NOs: 2, 3,4) were chosen based on these criteria.

Expression of reporter in glial cells by OLIG1-C promoter element. TheOLIG1-D DNA expression vector comprising the OLIG1 promoter elementcorresponding to SEQ ID NO: 1 (which is itself comprised of SEQ ID NOs:2, 3, 4 and 5) was introduced into mouse embryonic stem cells (ESCs) atthe HPRT locus. The ESCs were used to generate genetically modified micecontaining OLIG1-D. Immunohistochemical and immunofluorescence analysisof mouse brain tissue slices revealed EGFP expression throughout thecortical and subcortical regions of the brain, but not in the whitematter or corpus callosum (FIG. 3). Expression was observed inoligodendrocytes (FIG. 4).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

REFERENCES

-   Bronson, S. K., E. G. Plaehn, et al. (1996). “Single-copy transgenic    mice with chosen-site integration.”Proc Natl Acad Sci USA 93(17):    9067-72.-   Cheng, J., A. Dutra, et al. (2004). “Improved generation of C57BL/6J    mouse embryonic stem cells in a defined serum-free media.” Genesis    39(2): 100-4.-   Clapcote, S. J. and J. C. Roder (2005). “Simplex PCR assay for sex    determination in mice.” Biotechniques 38(5): 702, 704, 706.-   Hoang-Xuan, K., L. Aguirre-Cruz, et al. (2002). “OLIG-1 and 2 gene    expression and oligodendroglial tumours.” Neuropathol Appl Neurobiol    28(2): 89-94.-   Hogan, B., R. Beddington, et al. (1994). Manipulating the mouse.    Cold Spring Harbor, Cold Spring Harbor Laboratory Press.-   Hooper, M., K. Hardy, et al. (1987a). “HPRT-deficient (Lesch-Nyhan)    mouse embryos derived from germline colonization by cultured cells.”    Nature 326(6110): 292-5.-   Hooper, M., K. Hardy, et al. (1987b). “HPRT-deficient (Lesch-Nyhan)    mouse embryos derived from germline colonization by cultured cells.”    Nature 326: 292-295.-   Jasin, M., M. E. Moynahan, et al. (1996). “Targeted transgenesis.”    Proc Natl Acad Sci USA 93(17): 8804-8.-   Lee, K. H., C. K. Chuang, et al. (2007). “An alternative simple    method for mass production of chimeric embryos by coculturing    denuded embryos and embryonic stem cells in Eppendorf vials.”    Theriogenology 67(2): 228-37.-   Ligon, K. L., S. P. Fancy, et al. (2006). “Olig gene function in CNS    development and disease.” Glia 54(1): 1-10.-   Liu, L., E. E. Geisert, et al. (2007). “A transgenic mouse class-III    beta tubulin reporter using yellow fluorescent protein.” Genesis    45(9): 560-9.-   Ponchio, L., L. Duma, et al. (2000). “Mitomycin C as an alternative    to irradiation to inhibit the feeder layer growth in long-term    culture assays.” Cytotherapy 2(4): 281-6.-   van der Weyden, L., D. J. Adams, et al. (2002). “Tools for targeted    manipulation of the mouse genome.”Physiol Genomics 11(3): 133-64.-   Young, K. A., M. L. Berry, et al. (2002). “Fierce: a new mouse    deletion of Nr2e1; violent behaviour and ocular abnormalities are    background-dependent.” Behav Brain Res 132(2): 145-58.

1. An isolated polynucleotide comprising an OLIG1 regulatory elementoperably joined to an OLIG1 basal promoter through a non-native spacingbetween the promoter and the regulatory element.
 2. The isolatedpolynucleotide of claim 2, operably linked to an expressible sequence.3. A vector comprising the isolated polynucleotide of claim
 1. 4. Avector comprising the isolated polynucleotide of claim
 2. 5. A cellcomprising the vector of claim
 3. 6. The cell of claim 4, wherein thevector is stably integrated into the genome of the cell.
 7. The cell ofclaim 5, wherein the cell is a stem cell.
 8. A method of expressing asequence of interest, the method comprising operably linking thesequence of interest to the polynucleotide of claim 1; and introducinginto a cell permissive for expression from the OLIG1 promoter.